Project Summary Here we seek to develop a novel functional genomics tool, a RNA Polymerase II-dependent (RNAPII) eukaryotic cell-free expression system, that allows for rapid characterization of human regulatory components. There is a pressing need for new tools that can analyze the exponentially increasing amount of genomic data that has been collected through NHGRI sequencing initiatives and through advances in next-generation sequencing technology. Hidden within this genomic data is understanding to causes of human pathology. Understanding genomic data will become increasingly critical as new initiatives, such as TOPMed, seek to tie primary data to clinical outcomes. Synvitrobio?s technology produces cell-free systems that are able to express multiple DNA sequences without needing to grow cells. This allows for 100x higher-throughput analysis at 10-100x the time and cost of equivalent cellular expression. The cell-free system effectively prototypes a cell. They can be thought of as simplified and open environments to engineer biological complexity, while conserving the native cellular milieu of the originating source. The system takes in single or multiple DNA?s and conducts transcription and translation to produce functional protein. We propose developing a RNAPII-dependent eukaryotic cell-free expression system that is able to prototype a eukaryotic cell line. Current eukaryotic cell-free systems are able to conduct either native transcription or separately native translation, but not both simultaneously. Both native transcription and native translation are necessary for the end system to be capable of high-throughput functional genomics exploration. In the proposed Phase I feasibility effort, we will first demonstrate a HeLa combined nuclear and cytoplasmic cell-free expression system supporting RNAPII-dependent native transcription and native translation (Aim 1). We will also demonstrate two examples of gene regulation in a eukaryotic cell-free system: a RNAPII-independent example of operator/repressor binding, and a RNAPII-dependent example of allele-specific gene regulation (Aim 2). The long-term Phase II goal is to demonstrate the system?s ability to rapidly screen genomic data, going from bioinformatics collection, DNA synthesis, expression, and assay in a day, with a demonstration that collected results mirror expected results from cellular experiments.